Endothelial cells (EC) provide a semi-selective barrier between the blood and underlying tissue interstitium. Disruption of this barrier results in increased vascular permeability as seen in inflammation, tumor angiogenesis and atherosclerosis. Therefore, agents that enhance EC barrier function can be therapeutic for a variety of diseases. Sphingosine-1-phosphate (S1P) is a platelet-derived lipid that has many important effects on ECs including promoting increased barrier function that is dependent on S1P binding to its major cell surface receptor, S1P receptor, and activation of the small GTPase Rac1. This project proposes to elucidate the mechanism(s) of S1P regulation of Rac1 signaling, consequent downstream effectors and cortactin (an actin binding protein) translocation as they relate to cortical actin cytoskeletal rearrangement and increased endothelial cell barrier function. Specific Aim #1 focuses on characterization of S1P-mediated Rac1 activation in EC. Specifc Aim #2 focuses on mechanistically describing Rac-mediated cortical actin rearrangement after S1P treatment in EC. Specifc Aim #3 focuses on characterizing Rac-mediated cortactin protein interactions after S1P treatment in EC.